Methods and compositions for treatment of retinoid-responsive conditions

ABSTRACT

Methods and composition for treating a retinoid-responsive condition in a subject are provided according to embodiments of the present invention which include administering a therapeutically effective amount of a substance selected from the group consisting of: neutrophil gelatinase-associated lipocalin (NGAL), an NGAL analog, an NGAL stimulator and an analog of an NGAL stimulator, to a subject having a retinoid-responsive condition. Retinoid-responsive conditions illustratively include acne, rosacea, psoriasis, promyelocytic leukemia and neuroblastoma.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/393,331, filed Feb. 26, 2009, which claims priority from U.S.Provisional Patent Application Ser. Nos. 61/031,475, filed Feb. 26, 2008and 61/103,724, filed Oct. 8, 2008, the entire content of all of whichis incorporated herein by reference.

GOVERNMENT SPONSORSHIP

This invention was made with government support under RO1 AR47820awarded by NIAMS NIH. The government has certain rights in theinvention.

FIELD OF THE INVENTION

Described are compositions and methods generally relating to treatmentof retinoid-responsive conditions in a subject. In specific embodiments,compositions and methods described herein relate to neutrophilgelatinase-associated lipocalin, analogs and stimulators thereof, andtheir use in treatment of retinoid-responsive conditions in a subject.

BACKGROUND OF THE INVENTION

A number of pathological conditions, illustratively including acne,rosacea, psoriasis, promyelocytic leukemia and neuroblastoma, arecurrently treated using retinoids and are considered retinoid-responsiveconditions.

For example, acne is the most prevalent skin condition encountered bydermatologists, affecting nearly 85% of the people between the ages of12 and 24 years (15). While acne is not life-threatening, it does havesignificant physical and psychosocial morbidity (16). Acne results fromthe interplay of 4 factors: increased production sebum by the sebaceousgland, altered keratinization of follicular keratinocytes, activity ofPropionibacterium acnes (P. acnes) and inflammation. P. acnescontributes to the inflammation associated with acne via activation ofToll-like receptor 2 on the surface of inflammatory cells in the skininfiltrate (17).

Isotretinoin (13-cis RA) is the most potent agent that affects all thepathogenic features of acne. It is the only therapeutic agent thatdrastically reduces the size and secretion of sebaceous glands. However,isotretinoin, like thalidomide, is a teratogen and its use is closelyregulated through an FDA-mandated risk management program callediPLEDGE.

There is a continuing need for methods and compositions to treatretinoid-responsive conditions.

SUMMARY OF THE INVENTION

Methods of treating a retinoid-responsive condition in a subject areprovided according to embodiments of the present invention which includeadministering a therapeutically effective amount of a substance selectedfrom the group consisting of: neutrophil gelatinase-associated lipocalin(NGAL), an NGAL analog, an NGAL stimulator and an analog of an NGALstimulator, to a subject having a retinoid-responsive condition.Optionally, the NGAL analog is an NGAL receptor agonist. In a furtheroption, the NGAL stimulator is selected from the group consisting of: aToll-like receptor 2 ligand, a Toll-like receptor 3 ligand, an activatorof NFκB, leukotriene B4, cholesterol oleate and IL-17. Exemplaryactivators of NFκB that can be used in methods of treatment ofretinoid-responsive conditions include IL1-beta and TNF-alpha. IL1-betaand TNF-alpha can be used individually or in combination.

Retinoid-responsive conditions are well-known in the art and includeacne, rosacea, psoriasis, promyelocytic leukemia and neuroblastoma. Inembodiments of methods of the present invention, the protein isadministered to skin affected by: acne, rosacea and/or psoriasis. Infurther embodiments, the protein is administered to sebaceous glands ofa subject affected by: acne, rosacea and/or psoriasis.

An exemplary Toll-like receptor 2 ligand used in embodiments of methodsof the present invention isN-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine.An exemplary Toll-like receptor 3 ligand used in embodiments of methodsof the present invention is polyinosinic:polycytidylic acid.

Methods of treating a retinoid-responsive condition in a subjectaccording to embodiments of the present invention include stimulatingNGAL expression in a subject having a retinoid responsive condition.Methods of treating acne, rosacea, psoriasis, promyelocytic leukemia orneuroblastoma in a subject according to embodiments of the presentinvention include stimulating NGAL expression in a subject having acne,rosacea, psoriasis, promyelocytic leukemia or neuroblastoma. Inembodiments of methods of the present invention, NGAL expression isstimulated by administration of an NGAL stimulator which upregulatestranscription of a gene encoding NGAL and/or increases translation ofNGAL, to increase NGAL protein, to a subject having a retinoidresponsive condition. Optionally, NGAL expression is stimulated byadministration of a nucleic acid encoding NGAL to the subject.

An NGAL stimulator used in treatment of a retinoid-responsive conditionis selected from the group consisting of: a Toll-like receptor 2 ligand,a Toll-like receptor 3 ligand, an activator of NFκB, leukotriene B4,cholesterol oleate and IL-17 according to embodiments of methods of thepresent invention. In particular embodiments, the activator of NFκB isIL1-beta, TNF-alpha or a combination thereof. An exemplary Toll-likereceptor 2 ligand used in treatment of a retinoid-responsive conditionaccording to embodiments of the present invention isN-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine.An exemplary Toll-like receptor 3 ligand used in treatment of aretinoid-responsive condition according to embodiments of the presentinvention is polyinosinic:polycytidylic acid.

Methods of treating retinoid-responsive condition in a subject areprovided according to embodiments of the present invention which includeadministering a therapeutically effective amount of a protein selectedfrom: a protein comprising the amino acid of SEQ ID No. 1; a proteincomprising the amino acid of SEQ ID No. 2; a protein encoded by thecomplement of a nucleic acid that hybridizes under highly stringentconditions with the nucleotide sequence of SEQ ID No. 3; a proteinencoded by the complement of a nucleic acid that hybridizes under highlystringent conditions with the nucleotide sequence of SEQ ID No. 4;wherein the highly stringent conditions are: hybridization in a solutioncontaining 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes;a protein comprising an amino acid sequence that is at least 95%identical to SEQ ID No. 1; and a protein comprising an amino acidsequence that is at least 95% identical to SEQ ID No. 2, to a subjecthaving a retinoid-responsive condition.

Methods of inhibiting Propionibacterium acnes are provided according tomethods of the present invention which include contacting aPropionibacterium acnes bacterium with a substance selected from thegroup consisting of: NGAL and an NGAL analog. In preferred embodiments,the Propionibacterium acnes bacterium is in contact with a humansubject, such as a subject having acne, rosacea or a predisposition tohaving acne or rosacea.

In further embodiments, methods of inhibiting Propionibacterium acnesare provided according to the present invention which includeadministering an NGAL stimulator and/or an analog of an NGAL stimulatorto a subject colonized by Propionibacterium acnes or having apredisposition to colonization by Propionibacterium acnes.

Compositions are provided according to embodiments of the presentinvention which include neutrophil gelatinase-associated lipocalin(NGAL), an NGAL analog, an NGAL stimulator or an analog of an NGALstimulator. A combination of two or more of neutrophilgelatinase-associated lipocalin (NGAL), an NGAL analog, an NGALstimulator and an analog of an NGAL stimulator can be included incompositions according to embodiments of the present invention.Compositions according to embodiments of the present inventionoptionally include a pharmaceutically acceptable carrier. NGALstimulators included in compositions according to embodiments of thepresent invention can be a Toll-like receptor 2 ligand, a Toll-likereceptor 3 ligand, an activator of NFκB,N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[5]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine,polyinosinic:polycytidylic acid, leukotriene B4, cholesterol oleate orIL-17. A combination of two or more of a Toll-like receptor 2 ligand, aToll-like receptor 3 ligand, an activator of NFκB, leukotriene B4,N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine,polyinosinic:polycytidylic acid, cholesterol oleate or IL-17 can beincluded in compositions according to embodiments of the presentinvention. In further embodiments, compositions according to the presentinvention include a protein selected from: a protein comprising theamino acid of SEQ ID No. 1; a protein comprising the amino acid of SEQID No. 2; a protein encoded by the complement of a nucleic acid thathybridizes under highly stringent conditions with the nucleotidesequence of SEQ ID No. 3; a protein encoded by the complement of anucleic acid that hybridizes under highly stringent conditions with thenucleotide sequence of SEQ ID No. 4; wherein the highly stringentconditions are: hybridization in a solution containing 6×SSC,5×Denhardt's solution, 30% formamide, and 100 micrograms/ml denaturedsalmon sperm at 37° C. overnight followed by washing in a solution of0.1×SSC and 0.1% SDS at 60° C. for 15 minutes; a protein comprising anamino acid sequence that is at least 95% identical to SEQ ID No. 1; anda protein comprising an amino acid sequence that is at least 95%identical to SEQ ID No. 2, to a subject having a retinoid-responsivecondition.

In still further embodiments, compositions and methods according to thepresent invention include a protein selected from: a protein comprisingthe amino acid of SEQ ID No. 1; a protein comprising the amino acid ofSEQ ID No. 2; a protein encoded by the complement of a nucleic acid thathybridizes under highly stringent conditions with nucleotides 74-670 ofthe nucleotide sequence of SEQ ID No. 3; a protein encoded by thecomplement of a nucleic acid that hybridizes under highly stringentconditions with nucleotides 23-625 of the nucleotide sequence of SEQ IDNo. 4; wherein the highly stringent conditions are: hybridization in asolution containing 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes;a protein comprising an amino acid sequence that is at least 95%identical to SEQ ID No. 1; and a protein comprising an amino acidsequence that is at least 95% identical to SEQ ID No. 2, to a subjecthaving a retinoid-responsive condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a reproduction of images showing representative TUNEL stainedsections from Patient 4 taken at baseline and at one week ofisotretinoin treatment;

FIG. 2A is a graph showing comparison of array analysis and QPCR on RNAobtained from patient skin biopsies at baseline and one week of 13-cisRA treatment;

FIG. 2B is a graph showing comparison of array analysis and QPCR on RNAobtained from SEB-1 sebocytes incubated for 72 hours;

FIG. 3 is a reproduction of images showing immunostained sectionsindicating that NGAL (also termed lipocalin 2 and LCN2) expression isincreased in sebaceous glands in patients biopsied at one week ofisotretinoin treatment;

FIG. 4A is a set of two graphs showing expression of LCN2 mRNA detectedafter 48 hours of 13-cis RA (left panel) or ATRA (right panel)treatment;

FIG. 4B is a set of two graphs and corresponding immunoblots showingLCN2 protein expression after 72 hours of 13-cis RA (left panel) or ATRA(right panel) treatment;

FIG. 5A shows representative images of TUNEL assay results followingtreatment of SEB-1 sebocytes using purified recombinant human NGAL atvarious concentrations and indicating increasing apoptosis withincreasing dosage;

FIG. 5B is a graph showing quantification of the percentage of TUNELpositive stained SEB-1 sebocytes in each treatment group after 24 hoursof treatment with purified recombinant human NGAL at variousconcentrations;

FIG. 6A is a graph showing QPCR results demonstrating decreasedexpression of LCN2 mRNA following treatment of SEB-1 sebocytes withanti-LCN2 siRNA;

FIG. 6B is a reproduction of images of immunoblots showing that proteinlevels of NGAL were undetectable by western blotting after 48 and 72hours of 13-cis RA and anti-LCN2 siRNA treatment;

FIG. 7 is an immunoblot and corresponding graph showing that treatmentof cells with anti-LCN2 siRNA blocks the induction of caspase 3 cleavageby 13-cis retinoic acid in SEB-1 sebocytes;

FIG. 8A is a reproduction of images of SEB-1 sebocytes immunostainedusing an antibody to the murine 24p3 receptor (also known as the murinelipocalin 2 receptor and the murine NGAL receptor) showing presence ofthe receptor in cytoplasm of the cells;

FIG. 8B is a reproduction of an image of a Western blot analysisconfirming presence of the murine 24p3 receptor in SEB-1 sebocytes andindicating two receptor isoforms are present in SEB-1 sebocytes;

FIG. 9 is a graph showing that NGAL expression is significantlyincreased through Toll-like receptor 2 signaling;

FIG. 10 is a reproduction of images of immunoblots for NGAL indicatingthat NGAL expression increases in response to poly (I:C) treatment; and

FIG. 11 is a graph showing inhibitory effects of NGAL on P. acnesbacteria.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions relating to neutrophil gelatinase-associatedlipocalin (NGAL, also referred to as lipocalin 2 and LCN2), NGALanalogs, and use of NGAL and NGAL analogs, are provided according toembodiments of the present invention for treatment ofretinoid-responsive conditions. Methods and compositions relating toincreasing levels and/or activity of NGAL and/or NGAL analogs, and useof substances which increase levels and/or activity of NGAL and/or NGALanalogs, are provided according to embodiments of the present inventionfor treatment of retinoid-responsive conditions.

The term “retinoid-responsive condition” refers to any disease orcondition for which administration of one or more retinoids has abeneficial effect. Retinoid-responsive conditions that can be treatedusing compositions and methods of the present invention include, but arenot limited to, inflammatory dermatological conditions including, butnot limited to, acne, rosacea, urticaria, eczema and psoriasis; andproliferative disorders including, but not limited to, promyelocyticleukemia and neuroblastoma.

Retinoids are well-known in the art and include retinol, retinal,tretinoin, isotretinoin, alitretinoin, etretinate, acitretin,tazarotene, bexarotene and adapalene.

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; and B. Alberts et al.,Molecular Biology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson andM. M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman &Company, 2004.

NGAL is a protein which was initially isolated from neutrophils asdescribed in references 27-32.

The term “NGAL” refers to NGAL protein disclosed herein and orthologsfrom any species. The term “NGAL analog” refers to a substance having afunctional characteristic of NGAL. In particular, an NGAL analog iseffective to produce a desired physiologic or pharmacologic effect in asubject having a retinoid-responsive pathological condition, prevents orameliorates a retinoid-responsive pathological condition in the subject.In further embodiments, an NGAL analog is effective to inhibitPropionibacterium acnes.

NGAL used in methods according to embodiments of the present inventionand included in compositions according to embodiments of the presentinvention can be apo-NGAL or holo-NGAL.

In embodiments of the present invention the term “NGAL” refers toproteins of SEQ ID Nos. 1 and 2 and analogs thereof which have a desiredNGAL function, particularly inhibition of P. acnes and/or apoptoticactivity in sebocytes.

Human NGAL is disclosed herein as SEQ ID No. 1:

(SEQ ID No. 1) MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG 

Mouse NGAL is disclosed herein as SEQ ID No. 2:

(SEQ ID No. 2) MALSVMCLGLALLGVLQSQAQDSTQNLIPAPSLLTVPLQPDFRSDQFRGRWYVVGLAGNAVQKKTEGSFTMYSTIYELQENNSYNVTSILVRDQDQGCRYWIRTFVPSSRAGQFTLGNMHRYPQVQSYNVQVATTDYNQFAMVFFRKTSENKQYFKITLYGRTKELSPELKERFTRFAKSLGLKDDNIIFSVPTDQCIDN 

As used herein, the term “NGAL analog” refers to both naturallyoccurring variations of a given NGAL protein and recombinantly preparedmutations of a given NGAL protein, as well as functional fragmentsthereof.

The term “NGAL analog” refers to a protein characterized by an aminoacid sequence substantially similar to the amino acid sequence of humanNGAL (SEQ ID No. 1) or mouse NGAL (SEQ ID No. 2) and which hassubstantially similar functional properties compared to human NGAL ormouse NGAL. Substantially similar analogs of human NGAL or mouse NGALhave at least 80%, or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or greater, amino acid sequence identity to human NGAL ormouse NGAL. An NGAL analog can be encoded by a nucleic acid sequencehaving substantial similarity to nucleic acid sequences encoding humanNGAL and mouse NGAL disclosed herein. A nucleic acid sequence havingsubstantial similarity to a nucleic acid sequence encoding human NGAL ormouse NGAL has at least 70%, at least 75%, or at least 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, nucleic acidsequence identity to nucleic acid sequences encoding human NGAL or mouseNGAL.

In embodiments of the present invention, a substantially similar nucleicacid sequence is characterized as having a complementary nucleic acidsequence capable of hybridizing to a nucleic acid sequence encodinghuman NGAL or mouse NGAL under high stringency hybridization conditions.

The term “nucleic acid” as used herein refers to RNA or DNA moleculeshaving more than one nucleotide in any form including single-stranded,double-stranded, oligonucleotide or polynucleotide. The term “nucleotidesequence” is used to refer to the ordering of nucleotides in anoligonucleotide or polynucleotide in a single-stranded form of nucleicacid.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” to aspecified second nucleotide sequence. For example, a nucleotide sequencemay have 80%, 90%, or 100% complementarity to a specified secondnucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10nucleotides of a sequence are complementary to the specified secondnucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is100% complementary to the nucleotide sequence 5′-AGCT-3′. Further, thenucleotide sequence 3′-TCGA- is 100% complementary to a region of thenucleotide sequence 5′-TTAGCTGG-3′.

The terms “hybridization” and “hybridizes” refer to pairing and bindingof complementary nucleic acids. Hybridization occurs to varying extentsbetween two nucleic acids depending on factors such as the degree ofcomplementarity of the nucleic acids, the melting temperature, Tm, ofthe nucleic acids and the stringency of hybridization conditions, as iswell known in the art. The term “stringency of hybridization conditions”refers to conditions of temperature, ionic strength, and composition ofa hybridization medium with respect to particular common additives suchas formamide and Denhardt's solution. Determination of particularhybridization conditions relating to a specified nucleic acid is routineand is well known in the art, for instance, as described in J. Sambrookand D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., ShortProtocols in Molecular Biology, Current Protocols; 5th Ed., 2002. Highstringency hybridization conditions are those which only allowhybridization of substantially complementary nucleic acids. Typically,nucleic acids having about 85-100% complementarity are considered highlycomplementary and hybridize under high stringency conditions.Intermediate stringency conditions are exemplified by conditions underwhich nucleic acids having intermediate complementarity, about 50-84%complementarity, as well as those having a high degree ofcomplementarity, hybridize. In contrast, low stringency hybridizationconditions are those in which nucleic acids having a low degree ofcomplementarity hybridize.

The terms “specific hybridization” and “specifically hybridizes” referto hybridization of a particular nucleic acid to a target nucleic acidwithout substantial hybridization to nucleic acids other than the targetnucleic acid in a sample.

Stringency of hybridization and washing conditions depends on severalfactors, including the Tm of the probe and target and ionic strength ofthe hybridization and wash conditions, as is well-known to the skilledartisan. Hybridization and conditions to achieve a desired hybridizationstringency are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001;and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology,Wiley, 2002.

An example of high stringency hybridization conditions is hybridizationof nucleic acids over about 100 nucleotides in length in a solutioncontaining 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes.SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovineserum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone. Under highlystringent conditions, SEQ ID No. 3 or SEQ ID No. 4 will hybridize to thecomplement of substantially identical targets and not to unrelatedsequences. In preferred embodiments, under highly stringent conditions,at least the coding region of SEQ ID No. 3 (nucleotides 74-670) or SEQID No. 4 (nucleotides 23-625) will hybridize to the complement ofsubstantially identical targets and not to unrelated sequences.

Mutations can be introduced using standard molecular biology techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. One ofskill in the art will recognize that one or more amino acid mutationscan be introduced without altering the functional properties of NGAL.For example, one or more amino acid substitutions, additions, ordeletions can be made without altering the functional properties ofNGAL.

Conservative amino acid substitutions can be made in NGAL proteins toproduce NGAL analogs. Conservative amino acid substitutions are artrecognized substitutions of one amino acid for another amino acid havingsimilar characteristics. For example, each amino acid may be describedas having one or more of the following characteristics: electropositive,electronegative, aliphatic, aromatic, polar, hydrophobic andhydrophilic. A conservative substitution is a substitution of one aminoacid having a specified structural or functional characteristic foranother amino acid having the same characteristic. Acidic amino acidsinclude aspartate, glutamate; basic amino acids include histidine,lysine, arginine; aliphatic amino acids include isoleucine, leucine andvaline; aromatic amino acids include phenylalanine, glycine, tyrosineand tryptophan; polar amino acids include aspartate, glutamate,histidine, lysine, asparagine, glutamine, arginine, serine, threonineand tyrosine; and hydrophobic amino acids include alanine, cysteine,phenylalanine, glycine, isoleucine, leucine, methionine, proline, valineand tryptophan; and conservative substitutions include substitutionamong amino acids within each group. Amino acids may also be describedin terms of relative size, alanine, cysteine, aspartate, glycine,asparagine, proline, threonine, serine, valine, all typically consideredto be small.

NGAL analogs can include synthetic amino acid analogs, amino acidderivatives and/or non-standard amino acids, illustratively including,without limitation, alpha-aminobutyric acid, citrulline, canavanine,cyanoalanine, diaminobutyric acid, diaminopimelic acid,dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline,norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan,1-methylhistidine, 3-methylhistidine, and ornithine.

Embodiments of methods and compositions of the present invention includeNGAL proteins having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% amino acid sequence identity to SEQ ID No. 1 or SEQ ID No. 2.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:22642268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searchesare performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules of the present invention. BLAST proteinsearches are performed with the XBLAST program parameters set, e.g., toscore 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST are utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST is used to perform an iterated search which detects distantrelationships between molecules (Id.). When utilizing BLAST, GappedBLAST, and PSI Blast programs, the default parameters of the respectiveprograms (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBIwebsite). Another preferred, non limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, 1988, CABIOS 4:11 17. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 is used.

The percent identity between two sequences is determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

NGAL can be produced in recombinant host cells using well-knownconventional techniques. Broadly described, a nucleic acid moleculeencoding NGAL is operably linked to regulatory sequences that controltranscriptional expression in an expression vector. The expressionvector is introduced into a host cell where it is expressed and the NGALcan then be isolated.

An expression vector including a nucleic acid encoding NGAL isadministered to a subject to treat a retinoic-acid responsive conditionaccording to embodiments of the present invention.

NGAL of SEQ ID No. 1 is encoded by nucleotides 74-670 of the human NGALmRNA of SEQ ID No. 3:

  1 actcgccacc tcctcttcca cccctgccag gcccagcagc caccacagcg cctgcttcct 61 cggccctgaa atcatgcccc taggtctcct gtggctgggc ctagccctgt tgggggctct121 gcatgcccag gcccaggact ccacctcaga cctgatccca gccccacctc tgagcaaggt181 ccctctgcag cagaacttcc aggacaacca attccagggg aagtggtatg tggtaggcct241 ggcagggaat gcaattctca gagaagacaa agacccgcaa aagatgtatg ccaccatcta301 tgagctgaaa gaagacaaga gctacaatgt cacctccgtc ctgtttagga aaaagaagtg361 tgactactgg atcaggactt ttgttccagg ttgccagccc ggcgagttca cgctgggcaa421 cattaagagt taccctggat taacgagtta cctcgtccga gtggtgagca ccaactacaa481 ccagcatgct atggtgttct tcaagaaagt ttctcaaaac agggagtact tcaagatcac541 cctctacggg agaaccaagg agctgacttc ggaactaaag gagaacttca tccgcttctc601 caaatctctg ggcctccctg aaaaccacat cgtcttccct gtcccaatcg accagtgtat661 cgacggctga gtgcacaggt gccgccagct gccgcaccag cccgaacacc attgagggag721 ctgggagacc ctccccacag tgccacccat gcagctgctc cccaggccac cccgctgatg781 gagccccacc ttgtctgcta aataaacatg tgccctcagg ccaaaaaaaa aaaaaaaaaa

NGAL of SEQ ID No. 2 is encoded by nucleotides 23-625 of the mouse NGALmRNA of SEQ ID No. 4:

  1 agacctagta gctgtggaaa ccatggccct gagtgtcatg tgtctgggcc ttgccctgct 61 tggggtcctg cagagccagg cccaggactc aactcagaac ttgatccetg ccccatctct121 gctcactgtc cccctgcagc cagacttccg gagcgatcag ttccggggca ggtggtacgt181 tgtgggcctg gcaggcaatg cggtccagaa aaaaacagaa ggcagcttta cgatgtacag241 caccatctat gagctacaag agaacaatag ctacaatgtc acctccatcc tggtcaggga301 ccaggaccag ggctgtcgct actggatcag aacatttgtt ccaagctcca gggctggcca361 gttcactctg ggaaatatgc acaggtatcc tcaggtacag agctacaatg tgcaagtggc421 caccacggac tacaaccagt tcgccatggt atttttccga aagacttctg aaaacaagca481 atacttcaaa attaccctgt atggaagaac caaggagctg tcccctgaac tgaaggaacg541 tttcacccgc tttgccaagt ctctgggcct caaggacgac aacatcatct tctctgtccc601 caccgaccaa tgcattgaca actgaatggg tggtgagtgt ggctgactgg gatgcgcaga661 gacccaatgg ttcaggcgct gcctgtctgt ctgccactcc atctttcctg ttgccagaga721 gccacctggc tgccccacca gccaccatac caaggagcat ctggagcctc ttcttatttg781 gccagcactc cccatccacc tgtcttaaca ccaccaatgg cgtccccttt ctgctgaata841 aatacatgcc ccc

Non-limiting examples of regulatory sequences that controltranscriptional expression in an expression vector illustrativelyinclude a promoter, an enhancer, a splicing signal, a transcriptionstart site, a transcription termination signal, a polyadenylationsignal, an internal ribosome entry site (IRES) and combinations of theseor other regulatory sequences. A secretory sequence encoding a secretionsignal that directs an encoded heterologous protein into the secretorypathway of a host cell is optionally included. Additional sequencesoptionally included in an expression vector include one or moresequences encoding a marker suitable for selection of cells carrying theexpression vector.

Viral expression vectors can be used to express a desired protein.Non-limiting examples of virus expression systems include adenovirus,adeno-associated virus, herpes virus, vaccinia virus and lentivirus.

A host cell for expression of NGAL can be prokaryotic or eukaryotic,such as bacterial, plant, insect, fungus, yeast, and mammalian cells.

An expression vector is introduced into a host cell using well-knowntechniques such as infection or transfection, including calciumphosphate transfection, liposome-mediated transfection, electroporationand sonoporation. Expression constructs and methods for their generationand use to express a desired protein are known in the art, as described,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., (Eds.),Short Protocols in Molecular Biology, Wiley, 2002; and S. J. Higgins andB. D. Hames (Eds.), Protein Expression: A Practical Approach, OxfordUniversity Press, USA, 1999.

In addition to recombinant methodology, chemical synthetic techniquescan be used to produce NGAL. For example, NGAL can be produced usingsolid phase synthesis, solution phase synthesis, partial solid phasesynthesis or fragment condensation.

The term “isolated” as used herein refers to a substance that has beenseparated from contaminating cellular components associated with thesubstance in nature not intended to be associated with the substance andthat would interfere with use of the substance in therapeutic,prophylactic, diagnostic or other uses. Generally, an isolated substancedescribed herein is at least about 80% pure, at least about 90% pure, atleast about 95% pure, or greater than about 99% pure. Purification isachieved using well-known standard methodology such as fractionationand/or chromatography, such as ammonium sulfate precipitation andelution chromatography such as size exclusion chromatography,displacement chromatography, ion exchange chromatography and bioaffinitychromatography. Exemplary purification methodology is described in S.Doonan, Protein Purification Protocols Humana Press, 1996.

In embodiments of the present invention, isolated NGAL, an isolated NGALanalog, an isolated NGAL stimulator and/or an isolated analog of an NGALstimulator is administered to a subject and/or included in a compositionof the present invention.

A substance effective to increase NGAL and/or produce a therapeuticeffect of NGAL in a subject is administered to a subject having aretinoid-responsive condition according to embodiments of the presentinvention.

The term “NGAL stimulator” refers to substances effective to increaseNGAL in a subject, illustratively including expression vectors encodingNGAL, Toll-like receptor 2 (TLR2) ligands, Toll-like receptor 3 (TLR3)ligands, activators of NFkB, leukotriene B4, cholesterol oleate, andIL-17. The term “analog of an NGAL stimulator” refers to structuralvariants of NGAL stimulators that are functional to increase NGAL in asubject.

A substance effective to produce a therapeutic effect of NGAL in asubject includes an NGAL receptor agonist. The term “NGAL receptoragonist” refers to a substance that interacts with a NGAL receptor andenhances or increases a function of the NGAL receptor. The term “NGALreceptor agonist” encompasses both full and partial NGAL receptoragonists.

The term “Toll-like receptor 2 ligand” refers to a substance thatinteracts with a Toll-like receptor 2 and enhances or increases afunction of the Toll-like receptor 2. The term “Toll-like receptor 2ligand” encompasses both full and partial Toll-like receptor 2 agonists.

The term “Toll-like receptor 3 ligand” refers to a substance thatinteracts with a Toll-like receptor 3 and enhances or increases afunction of the Toll-like receptor 3. The term “Toll-like receptor 3ligand” encompasses both full and partial Toll-like receptor 3 agonists.

The term “activator of NFkB” refers to a substance that increasesactivity of NFkB to stimulate transcription of NGAL. Non-limitingexamples of substances that are activators of NFkB are IL1-beta andTNF-alpha.

In embodiments of methods and compositions of the present invention, astimulator of NGAL expression is an NGAL ligand, a TLR2 ligand, TLR3ligand, or NFkB activator. A combination of any two or more substancesselected from a TLR2 ligand, a TLR3 ligand, and a NFkB activator can beused in embodiments of methods and compositions of the presentinvention.

In embodiments of methods and compositions of the present invention, anNGAL ligand, a TLR2 ligand, TLR3 ligand, or NFkB activator is a smallmolecule stimulator of NGAL expression. Small molecule stimulators ofNGAL expression, such as NGAL ligands, TLR2 ligands, TLR3 ligands, andNFkB activators, are identified by screening of substances in an assayto determine an effect of a putative stimulator on NGAL expression. Forexample, naturally occurring or synthetic organic or inorganicsubstances having a molecular mass in the range of about 50-3000Daltons, more preferably having a molecular mass in the range of about100-2000 Daltons, and still more preferably having a molecular mass inthe range of about 300-700 Daltons. Exemplary assays to identify smallmolecule stimulators of NGAL expression include contacting a cell whichnaturally or recombinantly expresses an NGAL receptor, TLR2, TLR3,and/or NFkB, with one or more putative small molecule stimulators anddetecting a change in NGAL expression compared to untreated cells.Methods for detection of change in expression of a selected nucleic acidor protein are well-known in the art, illustratively including Northernblot, RNAse protection, RT-PCR and immunoassays. The term “stimulator ofexpression” refers to an increase in transcription and/or translationwhich increased levels and/or activity of NGAL.

The terms “treating” and “treatment” used to refer to treatment of aretinoid-responsive condition in a subject includes: preventing,inhibiting or ameliorating the retinoid-responsive condition in asubject, such as slowing progression of the condition and/or reducing orameliorating a sign or symptom of the condition.

A subject treated according to methods and using compositions of thepresent invention can be mammalian or non-mammalian. Humans arepreferred subjects treated according to methods and using compositionsof the present invention, and a mammalian subject can be any mammalincluding, but not limited to, a non-human primate; a rodent such as amouse, rat, or guinea pig; a domesticated pet such as a cat or dog; ahorse, cow, pig, sheep, goat, or rabbit. A non-mammalian subject can beany non-mammal including, but not limited to, fish; and a bird such as aduck, goose, chicken, or turkey.

In a particular embodiment of the present invention a method of treatingacne in a subject is provided which includes administering atherapeutically effective amount of NGAL, an NGAL analog, an NGALstimulator or an analog of an NGAL stimulator to a subject having acne.

A therapeutically effective amount is an amount which produces a desiredphysiologic or pharmacologic effect in a subject, prevents orameliorates a condition being treated in the subject. For example, atherapeutically effective amount is an amount which reduces oreliminates a sign or symptom of a retinoid-responsive condition beingtreated in the subject.

Suitable dosages ranges of NGAL, an NGAL analog, an NGAL stimulator oran analog of an NGAL stimulator depend on various factors such as theage of the subject, the severity and type of condition being treated inthe subject, the general condition of the subject, the route and form ofadministration of the composition being administered and the particularcomposition administered. One of ordinary skill in the art will be ableto ascertain a therapeutically effective amount without undueexperimentation in view of the present disclosure and what is known inthe art.

Administration of NGAL, an NGAL analog, an NGAL stimulator or an analogof an NGAL stimulator according to embodiments of a method of thepresent invention includes administration according to a dosage regimento produce a desired response. For example, one or more dosage units ofNGAL, an NGAL analog, an NGAL stimulator or an analog of an NGALstimulator is administered to a subject at one time in particularembodiments. A suitable schedule for administration of doses depends onseveral factors including age, weight, gender, medical history andhealth status of the subject, type of composition used and route ofadministration, for example. One of skill in the art is able to readilydetermine a dose and schedule of administration for a particularsubject.

Embodiments of the present invention optionally include administrationof a pharmacologically active agent in addition to NGAL, an NGAL analog,an NGAL stimulator or an analog of an NGAL stimulator. Non-limitingexamples of pharmacologically active agents administered in addition toNGAL, an NGAL analog, an NGAL stimulator or an analog of an NGALstimulator, in combination or separately, according to embodiments ofmethods of the present invention include, antibiotics, antivirals,antineoplastic agents, analgesics, antipyretics, antidepressants,antipsychotics, anticancer agents, antihistamines, anti-osteoporosisagents, anti-osteonecrosis agents, antiinflammatory agents, anxiolytics,chemotherapeutic agents, diuretics, growth factors, hormones,non-steroidal anti-inflammatory agents and vasoactive agents.

A pharmaceutically acceptable carrier can be included in a compositionincluding NGAL, an NGAL analog, an NGAL stimulator or an analog of anNGAL stimulator. A pharmaceutically acceptable carrier is substantiallynon-toxic to the subject in amounts administered and has substantiallyno deleterious effects on any active component of a composition in whichit is included.

The NGAL, an NGAL analog, an NGAL stimulator or an analog of an NGALstimulator is formulated for topical, local and/or systemicadministration to the subject.

Methods according to embodiments of the present invention includeadministration of NGAL, an NGAL analog, an NGAL stimulator or an analogof an NGAL stimulator as pharmaceutical formulations, including thosesuitable for oral, rectal, nasal, pulmonary, epidural, ocular, otic,intraarterial, intracardiac, intracerebroventricular, intracranial,intradermal, intravenous, intramuscular, intraperitoneal, intraosseous,intrathecal, intravesical, subcutaneous, topical, transdermal, andtransmucosal, such as by sublingual, buccal, vaginal, and inhalationalroutes of administration.

A pharmaceutical formulation of NGAL, an NGAL analog, an NGAL stimulatoror an analog of an NGAL stimulator according to embodiments of thepresent invention is in any dosage form suitable for administration to asubject, illustratively including solid, semi-solid and liquid dosageforms such as tablets, capsules, powders, granules, suppositories,pills, solutions, suspensions, ointments, lotions, creams, gels, pastes,sprays and aerosols.

Liposomes and emulsions are well-known types of pharmaceuticalformulations that can be used to deliver a pharmaceutical agent,particularly a hydrophobic pharmaceutical agent. In embodiments of thepresent invention, liposomes are particles typically produced as aunilammellar bilayer or a multilammellar bilayer of amphipathicmolecules enclosing an aqueous interior. Liposomes can include any typeof amphipathic materials compatible with a composition to be delivered,illustratively including naturally-occurring lipids, synthetic lipidsand combinations thereof.

A pharmaceutical formulation of a composition of the present inventioncan include a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” refers to a carrier which issuitable for use in a subject without undue toxicity or irritation tothe subject and which is compatible with other ingredients included in apharmaceutical composition. Pharmaceutically acceptable carriers,methods for making pharmaceutical compositions and various dosage forms,as well as modes of administration are well-known in the art, forexample as detailed in Pharmaceutical Dosage Forms: Tablets, eds. H. A.Lieberman et al., New York: Marcel Dekker, Inc., 1989; and in L. V.Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, 8th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins,2004; A. R. Gennaro, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins, 21st ed., 2005, particularly chapter 89;and J. G. Hardman et al., Goodman & Gilman's The Pharmacological Basisof Therapeutics, McGraw-Hill Professional, 10th ed., 2001.

A solid dosage form for administration or for suspension in a liquidprior to administration illustratively includes capsules, tablets,powders, and granules. In such solid dosage forms, one or more activeagents, is admixed with at least one carrier illustratively including abuffer such as, for example, sodium citrate or an alkali metal phosphateillustratively including sodium phosphates, potassium phosphates andcalcium phosphates; a filler such as, for example, starch, lactose,sucrose, glucose, mannitol, and silicic acid; a binder such as, forexample, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; a humectant such as, forexample, glycerol; a disintegrating agent such as, for example,agar-agar, calcium carbonate, plant starches such as potato or tapiocastarch, alginic acid, certain complex silicates, and sodium carbonate; asolution retarder such as, for example, paraffin; an absorptionaccelerator such as, for example, a quaternary ammonium compound; awetting agent such as, for example, cetyl alcohol, glycerolmonostearate, and a glycol; an adsorbent such as, for example, kaolinand bentonite; a lubricant such as, for example, talc, calcium stearate,magnesium stearate, a solid polyethylene glycol or sodium laurylsulfate; a preservative such as an antibacterial agent and an antifungalagent, including for example, sorbic acid, gentamycin and phenol; and astabilizer such as, for example, sucrose, EDTA, EGTA, and anantioxidant.

Solid dosage forms optionally include a coating such as an entericcoating. The enteric coating is typically a polymeric material.Preferred enteric coating materials have the characteristics of beingbioerodible, gradually hydrolyzable and/or gradually water-solublepolymers. The amount of coating material applied to a solid dosagegenerally dictates the time interval between ingestion and drug release.A coating is applied having a thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below 3associated with stomach acids, yet dissolves above pH 3 in the smallintestine environment. It is expected that any anionic polymerexhibiting a pH-dependent solubility profile is readily used as anenteric coating in the practice of the present invention to achievedelivery of the active agent to the lower gastrointestinal tract. Theselection of the specific enteric coating material depends on propertiessuch as resistance to disintegration in the stomach; impermeability togastric fluids and active agent diffusion while in the stomach; abilityto dissipate at the target intestine site; physical and chemicalstability during storage; non-toxicity; and ease of application.

Suitable enteric coating materials illustratively include cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,cellulose acetate, cellulose acetate phthalate, cellulose acetatetrimellitate, hydroxypropylmethyl cellulose phthalate,hydroxypropylmethyl cellulose succinate and carboxymethylcellulosesodium; acrylic acid polymers and copolymers, preferably formed fromacrylic acid, methacrylic acid, methyl acrylate, ammoniummethylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl; vinylpolymers and copolymers such as polyvinyl pyrrolidone, polyvinylacetate, polyvinylacetate phthalate, vinylacetate crotonic acidcopolymer, and ethylene-vinyl acetate copolymers; shellac; andcombinations thereof. A particular enteric coating material includesacrylic acid polymers and copolymers described for example U.S. Pat. No.6,136,345.

The enteric coating optionally contains a plasticizer to prevent theformation of pores and cracks that allow the penetration of the gastricfluids into the solid dosage form. Suitable plasticizers illustrativelyinclude, triethyl citrate (Citroflex 2), triacetin (glyceryltriacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400(polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, a coating composed of ananionic carboxylic acrylic polymer typically contains approximately 10%to 25% by weight of a plasticizer, particularly dibutyl phthalate,polyethylene glycol, triethyl citrate and triacetin. The coating canalso contain other coating excipients such as detackifiers, antifoamingagents, lubricants (e.g., magnesium stearate), and stabilizers (e.g.hydroxypropylcellulose, acids or bases) to solubilize or disperse thecoating material, and to improve coating performance and the coatedproduct.

Liquid dosage forms for oral administration include one or more activeagents and a pharmaceutically acceptable carrier formulated as anemulsion, solution, suspension, syrup, or elixir. A liquid dosage formof a composition of the present invention may include a colorant, astabilizer, a wetting agent, an emulsifying agent, a suspending agent, asweetener, a flavoring, or a perfuming agent.

For example, a composition for parenteral administration may beformulated as an injectable liquid. Examples of suitable aqueous andnonaqueous carriers include water, ethanol, polyols such as propyleneglycol, polyethylene glycol, glycerol, and the like, suitable mixturesthereof; vegetable oils such as olive oil; and injectable organic esterssuch as ethyloleate. Proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of a desirableparticle size in the case of dispersions, and/or by the use of asurfactant, such as sodium lauryl sulfate. A stabilizer is optionallyincluded such as, for example, sucrose, EDTA, EGTA, and an antioxidant.

For topical administration, a composition can be formulated foradministration to the skin such as for local effect, and/or as a “patch”formulation for transdermal delivery. Pharmaceutical formulationssuitable for topical administration include, for example, ointments,lotions, creams, gels, pastes, sprays and powders. Ointments, lotions,creams, gels and pastes can include, in addition to one or more activeagents, a base such as an absorption base, water-removable base,water-soluble base or oleaginous base and excipients such as athickening agent, a gelling agent, a colorant, a stabilizer, anemulsifying agent, a suspending agent, a sweetener, a flavoring, or aperfuming agent.

Transdermal formulations can include percutaneous absorption enhancerssuch as acetone, azone, dimethyl acetamide, dimethyl formamide, dimethylsulfoxide, ethanol, oleic acid, polyethylene glycol, propylene glycoland sodium lauryl sulfate. Iontophoresis and/or sonophoresis can be usedto enhance transdermal delivery.

Powders and sprays for topical administration of one or more activeagents can include excipients such as talc, lactose and one or moresilicic acids. Sprays can include a pharmaceutical propellant such as afluorinated hydrocarbon propellant, carbon dioxide, or a suitable gas.Alternatively, a spray can be delivered from a pump-style spray devicewhich does not require a propellant. A spray device delivers a metereddose of a composition contained therein, for example, using a valve forregulation of a delivered amount.

Opthalmic formulations of one or more active agents can includeingredients such as a preservative, a buffer and a thickening agent.

In particular embodiments of the present invention, an NGAL analog is anNGAL receptor agonist.

Further embodiments of the present invention include a method oftreating acne in a subject which includes stimulating NGAL expression ina subject having acne. For example, NGAL expression is stimulated byadministration of a transcription factor which upregulates transcriptionof a gene encoding NGAL and/or by administration of a nucleic acidencoding NGAL to the subject. Stimulators of NGAL expressionillustratively include leukotriene B4 and cholesterol oleate. IL-17 oractivators of NFκB is a further example of a stimulator of NGALexpression. Toll-like receptor 2 (TLR2) ligands and Toll-like receptor 3(TLR3) ligands are stimulators of NGAL expression and one or more TLR2ligands and/or one or more TLR3 ligands are administered to treat acnein a subject according to embodiments of the present invention.

Methods of inhibiting Propionibacterium acnes are provided byembodiments of the present invention which include administering NGAL,an NGAL analog, an NGAL stimulator or an analog of an NGAL stimulator toa subject colonized with Propionibacterium acnes. In particularembodiments of the present invention, administration includes contactinga Propionibacterium acnes bacterium with NGAL and/or a functional analogof NGAL. The skilled artisan will recognize that detecting P. acnescolonization in a subject and detecting P. acnes inhibition by methodsand compositions according to the present invention can be achievedusing well-known assay methods.

In preferred embodiments, the Propionibacterium acnes bacterium is in oron a human subject. The NGAL, an NGAL analog, an NGAL stimulator or ananalog of an NGAL stimulator is administered to the human subject by atopical, local and/or systemic route. Methods of inhibitingPropionibacterium acnes in a subject are useful in treatment ofconditions associated with Propionibacterium acnes colonization, such asacne and/or rosacea, for example.

In further embodiments of the present invention, methods of treating aretinoid-responsive condition in a subject are described which includeadministering a therapeutically effective amount of NGAL, an NGALanalog, an NGAL stimulator or an analog of an NGAL stimulator to asubject having a retinoid-responsive condition. Retinoid-responsiveconditions illustratively include acne, rosacea, psoriasis,promyelocytic leukemia and neuroblastoma.

In addition to NGAL, methods and compositions of the present inventionoptionally include methods and compositions relating to genes describedherein as regulated by isotretinoin, such as those listed in Tables IIand III.

A method of treating a retinoid-responsive condition in a subject isprovided according to embodiments of the present invention whichincludes administering a therapeutically effective amount of a Toll-likereceptor 2 ligand to a subject having a retinoid-responsive condition.

In particular embodiments, the therapeutically effective amount of aToll-like receptor 2 ligand is sufficient to increase NGAL and/or toincrease IL-8 in the subject.

In particular embodiments, the retinoid-responsive condition is acne.Optionally, the Toll-like receptor 2 ligand is administered to an areaof the skin of the subject affected by, or at risk of being affected by,acne.

Pam3CSK4, i.e.N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine×3HCl, is an example of a TLR2 agonist included in embodiments of methodsand compositions of the present invention.

A pharmaceutically acceptable carrier can be included in a compositionincluding a Toll-like receptor 2 ligand.

The Toll-like receptor 2 ligand is formulated for topical, local and/orsystemic administration to the subject according to particularembodiments of methods and compositions of the present invention.

A method of treating a retinoid-responsive condition in a subject isprovided according to embodiments of the present invention whichincludes administering a therapeutically effective amount of a Toll-likereceptor 3 ligand to a subject having a retinoid-responsive condition.

In particular embodiments, the therapeutically effective amount of aToll-like receptor 3 ligand is sufficient to increase NGAL in thesubject.

In particular embodiments, the retinoid-responsive condition is acne.Optionally, the Toll-like receptor 3 ligand is administered to an areaof the skin of the subject affected by, or at risk of being affected by,acne.

Polyinosinic:polycytidylic acid (Poly I:C) is an example of a TLR3agonist included in embodiments of methods and compositions of thepresent invention.

A pharmaceutically acceptable carrier can be included in a compositionincluding a Toll-like receptor 3 ligand.

The Toll-like receptor 3 ligand is formulated for topical, local and/orsystemic administration to the subject according to particularembodiments of methods and compositions of the present invention.

Methods of identifying NGAL analogs are provided by embodiments of thepresent invention which include administering a test compound to a cellsensitive to 13-cis retinoic acid-induced apoptosis to produce a treatedcell and assaying the treated cell for a marker of apoptosis and/orendogenous production of NGAL. A test compound which is a functionalanalog of NGAL is ineffective to induce the marker of apoptosis inabsence of a functional NGAL receptor in the treated cell in particularembodiments of inventive methods.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES Example 1 Patient Selection and Tissue Biopsies

Patients were recruited to have skin biopsies of their upper backs atbaseline and after one week of treatment with 13-cis RA. A total of 7patients that were prescribed isotretinoin by their dermatologist fortheir severe acne were enrolled in the study after signing informedconsent forms. Information regarding patient age, sex, the time ofbiopsy, and the dose of isotretinoin that patients were receiving at thetime of their second biopsy are presented in Table I.

TABLE I Isotretinoin Patient Demographic Patient Demographics DoseBiopsy Subject # Age Sex mg/kg/d (days) 1 15 M 0.5 7 2 17 M 0.5 7 3 17 M0.5 7 4 21 F 0.67 7 5 17 M 0.67 7 6 20 F 0.67 7 7 23 M 0.5 7 Mean ± SD18.5 ± 2.8 0.57 ± 0.09 7 ± 0

All protocols were approved by the Institutional Review Board of ThePennsylvania State University College of Medicine and were conductedaccording to the principles outlined in the Declaration of Helsinki. Allsubjects gave informed consent. Subjects included males and females ages14 to 40 years who were scheduled by their dermatologist to receivetreatment with 13-cis RA (isotretinoin, brand not noted) for severeacne. All aspects of the patients' treatment with 13-cis RA apart fromthe skin biopsies were standard of care and were not part of thisresearch. Exclusion criteria included patients with underlying medicalconditions requiring treatment with systemic medications that mightinterfere with the gene array analysis.

Seven patients had 5-mm punch biopsies of skin from the upper back takenat baseline and again at day 7 of treatment. Biopsies were taken fromnon-inflammed skin in areas apart from acne lesions. Biopsies wereplaced on ice and immediately transferred to the laboratory where theywere trimmed of fat and a small section of each biopsy was taken andparaffin-embedded for histology and immunohistochemistry. The remainingportion of the biopsy was flash frozen in liquid nitrogen and used fortotal RNA isolation.

Example 2

Histology suggests decrease in sebaceous gland volume after one week of13-cis RA treatment.

Hematoxylin and eosin staining was performed on sections of patient skinobtained from biopsies of skin from the upper back taken at baseline andat one week of treatment. A total of 17 sections at baseline and 19sections at one week taken from 6 patients were analyzed using imageanalysis software.

Images were captured using a Spot digital camera (DiagnosticInstruments, Inc.) and measurements were obtained with Image Pro PlusImaging Software version 3.0 after spatial calibration with a micrometerslide under the 10× objective. All areas of the sebaceous gland werecircled using a free-hand measuring tool and the total area of thesebaceous gland was calculated in each section from the baseline and theone-week biopsies. The average area for each distinct sebaceous glandwas calculated from sections analyzed. An ANOVA Single Factorstatistical test (α=0.05) was performed to look for significantdifferences in sebaceous gland area before and after one week oftreatment.

At baseline, sebaceous glands were characteristically large andmulti-lobular. Although changes in architecture were not obvious at oneweek of isotretinoin treatment, an early decrease in the overall size ofthe sebaceous glands was suggested. The mean area of sebaceous glands inthe baseline samples was 0.23±0.09 mm² (mean±SEM) compared to 0.12±0.02mm² in the one week samples, which was not significant using a pairedt-test (p=0.16) (data not shown).

Example 3 13-Cis RA Induces Apoptosis in Patient's Sebaceous Glands atOne Week

Skin sections were obtained from paraffin blocks of 7 patients sampledat baseline and one week of isotretinoin. A TUNEL-peroxidase assayfollowed by counter staining with hematoxylin was performed on skinbiopsies obtained from patients at baseline and at one week of 13-cis RAtherapy (n=6 pairs of samples).

In this procedure sections of baseline and post-treatment biopsies from7 patients were subjected to deparaffinization, rehydration andpermeabilization with 0.1% Triton-X, 0.1% sodium citrate in phosphatebuffered saline. Sections were subjected to In Situ Cell DeathDetection, Peroxidase kit (Roche Diagnostics, Indianapolis, Ind.)followed by counter staining with hematoxylin. DNase I treatment ofpositive and negative controls (without terminal transferase) wereincluded as assay controls according to manufacturer's instructions. Atleast 2 baseline and one week skin sections were analyzed from everypatient. Patient 7 was omitted from the analysis as no sebaceous glandswere found in baseline biopsy sections. Results were quantified bycounting positive stained cells/total cells in sebaceous glands. Datarepresents mean±SD, n=6 patients; paired t-test was used for statisticalanalysis and considered significant if p<0.05.

A 4-fold increase in the percentage of TUNEL positive cells was notedwithin the sebaceous gland at one week of therapy compared to baseline(45.9±11.9% vs. 13.9±6% TUNEL positive cells, p=0.005, paired t-test,α=0.05). FIG. 1 shows representative sections from Patient 4 taken atbaseline and at one week of isotretinoin treatment. NC=negative controlwhich consists of sections from a skin specimen treated with DNase I andprocessed without terminal transferase enzyme. PC=positive control whichconsists of sections from a skin specimen treated with DNase I andsubjected to the full TUNEL assay. Total magnification 400×.

TUNEL staining was strongest in the nuclei of sebocytes in the basallayer of the sebaceous gland and in early differentiated sebocytesadjacent to the basal layer, arrows. Apoptosis was selective for thesebaceous gland as no apoptosis was detected within the epidermis.

Example 4

Gene array expression analysis was performed on patient skin biopsies atbaseline and one week of isotretinoin therapy. Gene expression analysisof skin from patients treated with 13-cis RA revealed significantincreases in genes encoding calcium binding proteins, retinoid signalingmolecules, solute carriers and serine proteases.

In this procedure, total RNA was isolated from skin biopsies and DNasetreated using the RNeasy Fibrous Tissue Kit (Qiagen Inc., Valencia,Calif.). Approximately 2 μg of total RNA from each sample was used togenerate double stranded cDNA using a T7-oligo (dT) primer. BiotinylatedcRNA, produced through in vitro transcription, was fragmented andhybridized to an Affymetrix human U133A 2.0 microarray for one weekbiopsies. The arrays were processed on a GeneChip Fluidics Station 450and scanned on an Affymetrix GeneChip Scanner (Santa Clara, Calif.).Expression signals were normalized as previously described (47, 60, 61).Significant gene expression alterations were identified usingSignificance Analysis of Microarrays (SAM) computer software (62). SAMcontrols the false positives resulting from multiple comparisons throughcontrolling the false discovery rate (FDR) (63). FDR is defined as theresults of these array analyses have been submitted to the NationalCenter for Biotechnology proportion of false positive genes among allgenes that are considered significant. The Information Gene ExpressionOmnibus database under the accession number GSE10434. This is considereda super series and contains both the 1 week patient data and the cellculture model (SEB-1) data, described below. For the 1 week data alonethe accession number is GSE10432. For the SEB-1 data alone: GSE10433.

Array data was not generated for patient 3 due to insufficient quantityof RNA. Using a false discovery rate (FDR) of 0.05 that corresponds to a5% chance of false positive gene changes, 38 genes were significantlyup-regulated and 5 genes were significantly down-regulated by 13-cis RAwhen compared to baseline. (Table II; and Tables V and VI for a fulllisting of significantly changed genes). LCN2 was among the most highlyup-regulated genes.

TABLE II Significantly changed genes in patient skin after 1 week ofisotretinoin therapy Fold Gene Change Gene Title Symbol 7.03 lipocalin 2(oncogene 24p3) LCN2 6.2 S100 calcium binding protein A7 (psoriasin 1)S100A7 4.53 S100 calcium binding protein A9 (calgranulin B) S100A9 3.78solute carrier family 12 (K/Cl transporters) SLC12A8 3.32 cytochromeP450, family 2, subfamily B CYP2B7P1 2.61 serine (or cysteine)proteinase inhibitor SERPINA3 2.61 retinoic acid receptor responder(TIG 1) RARRES1 2.35 transmembrane protease, serine 4 TMPRSS4 1.99 S100calcium binding protein P S100P 1.92 ATPase, H+/K+ transporting,nongastric, alpha ATP12A 1.91 chemokine (C-C motif) ligand 2 CCL2 1.81retinol binding protein 1, cellular RBP1 1.69 solute carrier family 6(amino acid transporter) SLC6A14 1.67 E74-like factor 3 (ets domaintranscription factor) ELF3 1.56 cellular retinoic acid binding protein 2CRABP2 1.52 defensin, beta 1** DEFB1 1.51 calbindin 2, 29 kDa(calretinin) CALB2 1.5 S100 calcium binding protein A2 S100A2 1.49involucrin IVL 1.49 interleukin 27 receptor, alpha IL27RA −2.29 solutecarrier family 26, member 3 SLC26A3 −2.27 phospholipase A2, group VII(PAF acetyl- PLA2G7 hydrolase) −2.13 phosphodiesterase 6A,cGMP-specific, rod, alpha PDE6A

Example 5

Gene expression in SEB-1 sebocytes treated with 13-cis RA is similar toprofile the observed in human skin.

To identify sebocyte-specific gene changes induced by 13-cis RA, arrayanalysis was performed in SEB-1 sebocytes on three samples treated with0.1 micromolar 13-cis RA and three samples treated with vehicle.

The SEB-1 human sebocyte cell line was generated by transfection ofsecondary sebocytes with SV40 Large T antigen and was cultured instandard culture medium as previously described (59). 13-cis RA (R 3255)was purchased through SIGMA (St. Louis, Mo.). 10 mM Stock solutions of13-cis RA dissolved in ethanol were handled under dimmed yellow lightand stored under N₂ gas at −20° C. until use. Purified recombinant humanNGAL protein (amino acids 21-198) was purchased from R&D Systems(Minneapolis, Minn.) ready-to-use and was stored at −20° C. untilneeded. Stock solutions were diluted to desired concentrations instandard sebocyte culture medium.

Total RNA was isolated from SEB-1 sebocytes treated with 0.1 μM 13-cisRA or vehicle alone (0.001% ethanol) in three independent samples usingthe RNeasy kit (Qiagen Inc., Valencia, Calif.). Approximately 2 μg oftotal RNA from each sample was used to generate double stranded cDNAusing a T7-oligo (dT) primer. Biotinylated cRNA, produced through invitro transcription, was fragmented and hybridized to an U95Av2microarray for SEB-1 sebocytes. The arrays were processed on a GeneChipFluidics Station 450 and scanned on an Affymetrix GeneChip Scanner(Santa Clara, Calif.). Expression signals were normalized as previouslydescribed (47, 60, 61). Significant gene expression alterations wereidentified using Significance Analysis of Microarrays (SAM) computersoftware (62). SAM controls the false positives resulting from multiplecomparisons through controlling the false discovery rate (FDR) (63). FDRis defined as the proportion of false positive genes among all genesthat are considered significant.

A total of 85 genes (78 different genes) were significantly influencedby 13-cis RA: 58 were upregulated and 27 were down-regulated. Selectgenes whose expression was significantly changed genes are listed inTable III. (See Tables V and VI for the full listing of significantlychanged genes.) As in the patient samples, LCN2 and the tumorsuppressor, TIG1, demonstrated the greatest changes in gene expression.

TABLE III Selected significantly changed gene in SEB-1 sebocytes after13-cis RA treatment Fold Gene Change Gene Title Symbol 12.25 retinoicacid receptor responder (tazarotene RARRES1 induced) 1 7.04 lipocalin 2(oncogene 24p3) LCN2 5.95 tumor necrosis factor, alpha-induced protein 2TNFAIP2 5.91 hydroxyprostaglandin dehydrogenase 15-(NAD) HPGD 4.64cytochrome P450, family 1, subfamily B, poly- CYP1B1 peptide 1 4.18tumor necrosis factor (ligand) superfamily, TNFSF10 member 10 3.70serine (or cysteine) proteinase inhibitor, SERPINB3 3.65 homeo box A5HOXA5 3.43 insulin-like growth factor binding protein 3 IGFBP3 3.29aldehyde dehydrogenase 1 family, member A3 ALDH1A3 3.22 retinoic acidreceptor responder (tazarotene RARRES3 induced) 3 3.08 oxidised lowdensity lipoprotein (lectin-like) OLR1 receptor 1 2.60 cyclin-dependentkinase inhibitor 1A (p21, Cip1) CDKN1A 2.51 E74-like factor 3 (etsdomain transcription factor, ELF3 epithelial-specific) 2.42 interferonregulatory factor 1 IRF1 2.42 interferon-induced protein withtetratricopeptide IFIT3 repeats 3 2.09 proteasome (prosome, macropain)subunit, beta PSMB10 type, 10 2.08 vascular cell adhesion molecule 1VCAM1 2.07 annexin A9 ANXA9 2.07 2′,5′-oligoadenylate synthetase 1,40/46 kDa OAS1 1.85 GATA binding protein 3 GATA3 1.79 protein kinase C,alpha PRKCA 1.70 nuclear factor of kappa light polypeptide gene NFKB2enhancer in B-cells 1.69 Fas (TNF receptor superfamily, member 6) FAS−4.68 keratin 6A /// keratin 6C KRT6A −3.94 FK506 binding protein 5FKBP5 −3.25 ELOVL family member 5, elongation of long ELOVL5 chain fattyacids

Example 6 QPCR Verification of Select Genes from Array Analyses

QPCR analyses showed that the direction and magnitude of the change inexpression for the selected genes were similar to those observed withthe gene array analyses and verified siRNA knockdown and immunoblottingexperiments.

Applied Biosystems' Assays-on-Demand Taqman Universal PCR Master Mix,primer/probe sets, and ABI's 7900HT Fast Real-Time PCR System with384-well plate block module were used according to manufacturer'sinstructions (Applied Biosystems, Foster City, Calif.). Integrity ofisolated RNA was verified by agarose gel electrophoresis. cDNA wasgenerated from 1 μg of total RNA, primed with oligo-dT, using theSuperscript First-Strand Synthesis System for RT-PCR (Invitrogen,Carlsbad, Calif.). Diluted cDNA from the one week samples were run forthe reference gene TATA binding protein (TBP) and genes of interest:lipocalin 2, (LCN2) RARRES1, S100A7, SERPINA3 and PLA2G7. For gene arrayverification, 8 independent samples of SEB-1 sebocytes treated with 0.1μM 13-cis RA or vehicle alone for 72 hours were analyzed. Genes ofinterest included: LCN2, RARRES1, insulin-like growth factor bindingprotein 3 (IGFBP-3), GATA3, ZBTB16 and potential LCN2 cell surfacereceptor (SLC22A17). Expression of TBP, GAPDH and LCN2 was analyzed in 6independent SEB-1 samples for the siRNA knockdown experiments. SEB-1sebocytes treated with increasing concentrations of ATRA (n=5) wereanalyzed for the reference gene, TBP, and LCN2. In all experiments,assay controls included samples omitting reverse transcriptase enzyme aswell as samples without cDNA. Data was analyzed using the RelativeExpression Software Tool (REST-XL version 1) software program (64) withefficiency correction and a p-value <0.05 was considered significant.

Sufficient RNA was available from 5 of 7 patients (all but Patients 3and 4) to verify gene expression changes by QPCR for LCN2, retinoic acidreceptor responder 1 (RARRES1, TIG1), psoriasin (S100A7), serineprotease inhibitor A3 (SERPINA3) and phospholipase A2 group 7 (plateletactivating factor acetyl hydrolase, PLA2G7). FIG. 2A shows comparison ofarray analysis and QPCR on RNA obtained from patient skin biopsies atbaseline and one week of 13-cis RA treatment. Data represent themean±SEM of the fold-change in gene expression as determined by QPCR in5 subjects compared to array analysis performed in 6 subjects.

For SEB-1 sebocytes, gene array expression changes were verified by QPCRfor LCN2, TIG1, insulin-like growth factor binding protein 3 (IGFBP3),GATA transcription factor 3 (GATA3) and ZBTB16; zinc finger and BTBdomain-containing 16. FIG. 2B shows comparison of array analysis andQPCR on RNA obtained from SEB-1 sebocytes incubated for 72 hours in thepresence or absence of 13-cis RA. Data represent the mean±SEM of thefold change in gene expression as determined by QPCR in 8 samplescompared to array analysis performed in 3 samples. QPCR results wereanalyzed by REST-XL software program and *p<0.05 was consideredsignificant.

Example 7 13-Cis RA Induces Expression of Neutrophil GelatinaseAssociated Lipocalin (NGAL) in Patients' Sebaceous Glands

Patient skin biopsies that were taken at baseline and at one week ofisotretinoin therapy were used to assess NGAL expression andlocalization using immunohistochemistry.

FIG. 3 shows that NGAL expression is increased in sebaceous glands inpatients biopsied at one week of isotretinoin treatment.Immunohistochemistry using an antibody to NGAL was performed on skinsections taken at baseline and at one week of isotretinoin treatment.Sections were incubated overnight with a 1:50 dilution of mousemonoclonal lipocalin 2/NGAL antibody and developed using AEC chromagen(dark areas). All sections were counterstained with hematoxylin.Representative images at baseline and after 1 week isotretinoin fromPatients 1 and 2 are shown. An image of the epidermis after isotretinointreatment from Patient 2 is shown. NC=negative control, consists of anormal human skin incubated with normal mouse IgG₁ antibody. Originalmagnification: 100×. NGAL was expressed in the sebaceous gland and ductof samples of skin taken at one week of isotretinoin therapy. NGAL wasnot expressed in the epidermis.

In each patient, NGAL expression was detected specifically within thebasal layer of the sebaceous gland and upper sebaceous duct in sectionsfrom skin taken at one week of therapy as shown in FIG. 3. NGALexpression was not noted within the epidermis. This pattern of NGALexpression is comparable to the pattern of TUNEL positive cells afterisotretinoin treatment. NGAL immunoreactivity and TUNEL positive cellsare both present within the basal layer of the sebaceous gland and areabsent from the epidermis.

In general, NGAL expression is increased after one week of isotretinointreatment although, the amount of increase varied among the patients asshown in Table IV. Patient 7 was omitted from the analysis because nosebaceous glands were captured in the baseline biopsy sections. In 2 outof the 6 patients (Patients 4 and 6, the only females in the study), aminimal increase in NGAL staining was noted. Of the 6 patients examined,only one had NGAL expression in sebaceous glands at baseline, see TableIV.

TABLE IV Quantification of NGAL staining for individual patients atbaseline and after one week isotretinoin therapy. Baseline 1-week (% ofsebaceous gland (% of sebaceous gland Patient positive for NGAL)positive for NGAL) 1 0.0 36.7 2 0.0 11.3 3 0.0 27.1 4 0.0 1.1 5 11.112.1 6 0.0 1.0 Average 1.9 14.9 SEM 1.9 5.9

Example 8

The expression of NGAL within SEB-1 sebocytes was verified by QPCR andwestern blotting showing that 13-cis RA and ATRA increase LCN2 mRNA andNGAL protein expression in SEB-1 sebocytes.

LCN2 mRNA expression was verified by QPCR after 48 hours of retinoidtreatment. Data represent mean±SEM of the fold-change in gene expressionas determined by QPCR of 5 independent samples. Statistical analysis wasperformed with REST-XL software program and considered significant if*p<0.05.

For Western blotting, mouse monoclonal antibody to lipocalin 2 (NGAL)was obtained from Abcam, Inc., (Cambridge, Mass.). The 24p3R (NGALreceptor) affinity purified antibody, positive (HEK 293 cell lysate) andnegative (T47D cell lysate) controls were kindly provided by Dr. MichaelGreen (University of Massachusetts Medical School). GAPDH (#2118),Cleaved caspase 3 (#9664), β-actin (#4967) and anti-rabbit horseradishperoxidase (HRP) linked secondary antibodies (#7074) were purchased fromCell Signaling Technology (Beverly, Mass.). Secondary Anti-mouse HRPantibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz,Calif.).

SEB-1 sebocytes (passages 22-28) were in standard culture medium until50-75% confluent. Treatments of 13-cis RA (0.1 nM, 1 nM, 10 nM, 0.1 μM,or 1 μM,) or ethanol vehicle (0.01% or less) were applied for 48 or 72hours. In parallel studies, identical concentrations of all-transretinoic acid (ATRA) were applied for 72 hours. In all experiments, eachplate was considered an independent sample and total protein wascollected. Blots were incubated with appropriate primary and secondaryantibodies followed by densitometry. Each experiment was repeated aminimum of 3 independent times. Data was analyzed using a paired t-testand results were considered significant if p<0.05.

As shown in FIG. 4A, expression of LCN2 mRNA was detected after 48 hoursof 13-cis RA treatment. 13-cis RA concentrations of 1 nM and greatersignificantly increased LCN2 mRNA levels to approximately 6-fold withmicromolar concentrations when compared to control.

Protein expression was verified by western blot at 72 hours of retinoidtreatment. Blots were incubated with primary antibody to NGAL as well asβ-actin as a loading control. Blots were analyzed by densitometry andnormalized to β-actin. The graph represents normalized fold-changevalues (mean±SEM) relative to control for a minimum of 3 independentblots. Statistical analysis was performed with paired t-test, *p<0.05.As shown in FIG. 4B, after 72 hours of treatment with concentrationsranging from 0.1 nM to 1 μM 13-cis RA, NGAL protein expression increasedin a dose-dependent manner to a maximum of approximately 10-fold withmicromolar concentrations in SEB-1 sebocytes when compared to control(p<0.05).

The first 1000 base pairs of the LCN2 promoter were scanned for retinoicacid response elements (RAREs) using the predefined consensus sequenceswithin the Transfac database through the Transcription Element SearchSystem (TESS) and reveals the presence of both RAR and RXR bindingsites. Prior studies suggest that 13-cis RA acts as a reservoir of ATRAand that after isomerization to ATRA, acts via binding nuclear retinoicacid receptors (12). Although, in other studies, retinoidreceptor-independent modulation of signaling pathways by ATRA, such asbinding to PKC, have also been reported (13, 14).

To address this issue, SEB-1 sebocytes were treated with increasingconcentrations of ATRA in parallel studies and examined for expressionof LCN2 mRNA and NGAL protein. QPCR and immunoblotting show that NGALexpression also increased in a dose-dependent manner with ATRA whencompared to control. The level of NGAL expression induced by ATRA isless than that with 13-cis RA (7-fold vs. 10-fold) but the differencebetween ATRA and 13-cis RA was not significant, see FIGS. 4A and 4B.

Example 9 Recombinant NGAL Protein Induces Apoptosis in SEB-1 Sebocytes

SEB-1 sebocytes were treated with increasing concentrations of purifiedrecombinant human NGAL (rhNGAL) protein and a TUNEL assay was performed.

For the TUNEL assay, SEB-1 sebocytes (passage 22-28) were cultured in12-well plates in standard medium until approximately 30-40% confluent.Wells were rinsed with phosphate buffered saline (PBS) and treated induplicate with vehicle control, 1 pg/mL, 10 pg/mL, 100 pg/mL, 1 ng/mL,or 10 ng/mL of recombinant human NGAL (R&D Systems, Minneapolis, Minn.)or human actin (negative control; Cytoskeleton, Denver, Colo.) proteinfor 24 hours. In all experiments, each well was considered one sample.Samples were prepared by manufacturer's instructions for In Situ CellDeath Detection Kit, Fluorescein (Roche Diagnostics, Indianapolis, Ind.DNase I treatment of positive and negative controls (without terminaltransferase) were included as assay controls according to manufacturer'sinstructions. Results were analyzed and quantified by counting positivestained cells per total number of cells in 3 representative fields perwell for each of the treatments done in duplicate. Four independentrhNGAL experiments were performed whereas treatment with actin wasperformed once. Statistical analyses were performed between control andeach treatment concentration using ANOVA Two Factor with Replication andconsidered significant if p<0.05.

As noted above, SEB-1 sebocytes were treated with vehicle as a control,1 pg/mL, 10 pg/mL, 100 pg/mL, 1 ng/mL or 10 ng/mL of purifiedrecombinant human NGAL protein (R&D Systems) or the same concentrationsof human actin protein for 24 hours. After 24 hours of treatment withNGAL, the percentage of TUNEL positive cells in SEB-1 sebocytes wassignificantly increased to a maximum of approximately 35% using 1 ng/mLrhNGAL as shown in FIGS. 5A and 5B.

FIG. 5A shows representative images of the TUNEL assay using rhNGAL areshown. Original magnification: 200×. FIG. 5B shows quantification of thepercentage of TUNEL positive stained cells per treatment at 24 hours.Data represent mean±SEM, n=4-8. Parallel experiments to control fornonspecific effects of protein were performed using human actin protein(n=2). The percentage of TUNEL positive cells is less than 5% with allconcentrations of actin, which is similar to control values. Statisticalanalyses were performed between vehicle control and each treatmentconcentration of rhNGAL using ANOVA Two Factor with Replication;*p<0.05. rhNGAL significantly increased TUNEL staining compared tocontrol over a wide range of concentrations with maximal induction notedat 1 ng/mL.

Example 10 NGAL Mediates 13-Cis RA-Induced Apoptosis in Human Sebocytes

To test the hypothesis that NGAL mediates the apoptotic effect of 13-cisRA on sebocytes, siRNA to LCN2 is utilized in the presence of 13-cis RAand the effect on apoptosis using western blotting for cleaved caspase3, the activated form of caspase 3 is examined.

Optimization of the appropriate nucleofection solution and program wascarried out according to manufacturer's instructions using the Cell LineOptimization Nucleofector Kit with 2 μg of pgmaxGFP DNA in combinationwith the highest efficiency (GFP expression) and lowest mortality.Program T-20 with solution T was chosen for future experiments.Efficiency of nucleofection was determined by GFP expression quantifiedby flow cytometry with mock-nucleofected SEB-1 sebocyte controls. Veryhigh levels of GFP expression (87%, 90%, 73%, and 57%) were detected at24, 48, 72 and 96 hours post-nucleofection, respectively.

Using ON-TARGETplus Smartpool Human LCN2, Human GAPDH and siCONTROLsiRNA duplex oligonucleotides (Dharmacon Research, Lafayette, Colo.),nucleofection was performed as suggested by Dharmacon and AmaxaBiosystems. SEB-1 sebocytes, 2×10⁶ cells per 100 microliter reactionwere nucleofected with 1 μg siCONTROL, GAPDH or LCN2 siRNA. 13-cis RA(0.1 μM) was added 24 hours post-nucleofection. Extent of siRNAknockdown of gene expression was verified by QPCR and western blottingfor LCN2/NGAL and GAPDH at various time points after 13-cis RAtreatment. Involvement of LCN2/NGAL in mediating 13-cis RA inducedapoptosis was assessed by cleaved caspase 3 protein expression usingwestern blotting.

QPCR results shows in FIG. 6A demonstrate that the expression of LCN2mRNA was successfully decreased 15-fold (93%) by the siRNA compared tosiCONTROL in SEB-1 cells that were treated for 48 hours with 13-cis RA.FIG. 6A shows QPCR analysis of LCN2 and GAPDH mRNA levels at 48 hours of0.1 μM 13-cis RA treatment. The expression of LCN2 mRNA was successfullydecreased 15-fold by the LCN2 siRNA compared to siCONTROL whereasexpression of GAPDH was minimally affected by siRNA to LCN2. GAPDH mRNAexpression was decreased 4-fold by the specific GAPDH siRNA whencompared to siCONTROL whereas siRNA to GAPDH had minimal effects onexpression of mRNA for LCN2. Data represent mean±SEM; n=6. *p<0.05 asdetermined by REST-XL program. As a control, the specificity of siRNAknockdown was verified using siRNA to GAPDH in parallel samples. GAPDHand LCN2 gene expression were successfully inhibited in their respectivesamples.

FIG. 6B shows that protein levels of NGAL were undetectable by westernblotting after 48 and 72 hours of 13-cis RA treatment. FIG. 6B displaysimmunoblot analysis of GAPDH and NGAL protein levels: NGAL proteinexpression is decreased to undetectable levels by western blotting at 48and 72 hours of 0.1 μM 13-cis RA treatment with LCN2 siRNA.Representative blots of 4 independent experiments are shown.

After confirming successful inhibition of LCN2 and NGAL expression bysiRNA, the effect of LCN2 on apoptosis was determined.

Under siRNA conditions described for FIG. 6, immunoblotting revealedthat siRNA to LCN2 decreases expression of cleaved caspase 3 byapproximately 5-fold compared to siRNA control. siRNA to LCN2 in thepresence of 13-cis RA decreased expression of cleaved caspase 3 toapproximately 20% of control levels, corresponding to a 5-foldreduction, as indicated in FIG. 7.

Blots in FIG. 7 were incubated with primary antibodies to cleavedcaspase 3 as well as β-actin as a loading control to normalizedensitometry values. Three independent “scrambled siCONTROL (SCRAM)” and“LCN2” western blots are shown. p17 and p19 are the cleaved or “active”fragments of caspase 3. The graph in FIG. 7 represents the normalizedexpression of cleaved caspase 3 from 5 independent western blots;statistical analysis was performed with paired t-test, mean±SEM *p<0.05.These data indicate that NGAL mediates the apoptotic response of SEB-1sebocytes to 13-cis RA.

Example 11 SEB-1 Sebocytes Express the Receptor for NGAL

A cell surface receptor for the murine homolog to NGAL, 24p3, wasidentified in murine pro-B lymphocytic FL5.12 cells. The presence ofthis receptor (24p3R) is believed to be responsible for cell-specificsusceptibility to apoptosis (6). Based on sequence homology, the humanhomolog of 24p3R is predicted to be the solute carrier member, SLC22A17(6). Expression of SLC22A17 mRNA was detected in SEB-1 sebocytes by QPCRanalysis (data not shown).

24p3R (NGAL receptor) affinity purified antibody (6) was used. SEB-1sebocytes were cultured and fixed according to standard proceduresfollowed by overnight incubation with 24p3R antibody or normal rabbitIgG (negative control) and counterstaining with hematoxylin.

The 24p3/NGAL receptor was detected in SEB-1 sebocytes byimmunohistochemistry and western blotting using an antibody to the mouse24p3R(6).

FIGS. 8A and 8B show that SEB-1 sebocytes express the receptor for NGAL.FIG. 8A shows results of experiments in which SEB-1 sebocytes were grownunder standard conditions and immunocytochemistry was performed using anantibody to the murine 24p3 receptor. Slides were counterstained withhematoxylin. Negative control (NC) was processed with normal rabbit IgGantibody in place of the primary antibody. Magnification bar indicates50 μm. Immunoreactivity for the 24p3 receptor localizes to the cytoplasmof SEB-1 sebocytes.

Western analysis confirms presence of the receptor and indicates tworeceptor isoforms are present in SEB-1 sebocytes: high molecular weight(H.M.W.) and 24p3R long (24p3R-L). Positive ((+); HEK 293 cell lysate)and negative ((−); T47D cell lysate) controls are shown in FIG. 8B. Allsamples for this blot were run on the same gel but were noncontiguous.Blot shown is representative of 3 independent experiments.

Example 12

P. acnes induces NGAL expression through TLR2 signaling.

SEB-1 sebocytes are pretreated with TLR2 neutralizing antibody, orcorresponding isotype control antibody (mIgG2b) for one hour prior totreatment with 30 μg/mL P. acnes, vehicle or with TLR2 antibody alonefor 24 hours. An ELISA assay for NGAL is performed on culture medium andthe cell lysates. Data is analyzed using a paired Student's t-test n=4*p<0.05. FIG. 9 is a graph showing that, in the presence of P. acnes,NGAL expression is significantly increased in both the culture mediumand within cells when compared to vehicle and that, in the presence ofTLR2 neutralizing antibody, P. acnes induced NGAL expression decreases;close to vehicle levels.

Example 13

NGAL expression increases in response to poly (I:C) treatment in SEB-1sebocytes. SEB-1 sebocytes are treated with 20 μg/mL Poly (I:C),commercially available from Invivogen, San Diego, Calif., for 48 hrs.Total cell lysate is examined for NGAL expression by immunoblotting.Samples are run on same gel. FIG. 10 shows immunoblots for NGAL and thecontrol, beta-actin, indicating that NGAL expression increases inresponse to poly (I:C) treatment in SEB-1 sebocytes.

Example 14

P. acnes bacterium is treated with varying concentrations ofrecombinantly produced human NGAL (rhNGAL) produced in E. coli (1-25μg/mL) for 4 hours. Dilutions are plated and the percent survival iscalculated relative to vehicle. Both apo-NGAL (not bound to iron) andholo-NGAL (iron bound) are produced in E. coli and are easilydistinguishable from one another due to the fact that holo-NGAL has pinkhue when purified and apo-NGAL does not.

TABLE V Significantly Changed genes after one-week Isotretinointreatment. Probe ID Accession # old Δ Gene Title Symbol 212531_atNM_005564 .03 lipocalin 2 (oncogene 24p3) LCN2 205916_at NM_002963 .20S100 calcium binding protein A7 (psoriasin 1) S100A7 203535_at NM_002965.53 S100 calcium binding protein A9 (calgranulin B) S100A9 219874_atNM_024628 .78 solute carrier family 12 (K/Cl transporters) SLC12A8206754_s_at NM_000767 .32 cytochrome P450, family 2, subfamily BCYP2B7P1 202376_at NM_001085 .61 serine (or cysteine) proteinaseinhibitor SERPINA3 206392_s_at NM_002888 .61 retinoic acid receptorresponder (TIG 1) RARRES1 221872_at AI669229 .45 retinoic acid receptorresponder (TIG 1) RARRES1 206391_at NM_002888 .42 retinoic acid receptorresponder (TIG 1) RARRES1 218960_at NM_016425 .35 transmembraneprotease, serine 4 TMPRSS4 206478_at NM_014792 .27 KIAA0125 KIAA0125219014_at NM_016619 .21 placenta-specific 8 PLAC8 219554_at NM_016321.08 Rhesus blood group, C glycoprotein RHCG 221605_s_at AF136970 .04pipecolic acid oxidase PIPOX 204351_at NM_005980 .99 S100 calciumbinding protein P S100P 218498_s_at NM_014584 .96 ERO1-like (S.cerevisiae) ERO1L 207367_at NM_001676 .92 ATPase, H+/K+ transporting,nongastric, alpha ATP12A 216598_s_at S69738 .91 chemokine (C-C motif)ligand 2 CCL2 203423_at NM_002899 .81 retinol binding protein 1,cellular RBP1 219795_at NM_007231 .69 solute carrier family 6 (aminoacid transporter) SLC6A14 210827_s_at U73844 .67 E74-like factor 3 (etsdomain transcription factor) ELF3 221701_s_at AF352728 .62 stimulated byretinoic acid gene 6 homolog STRA6 204541_at NM_012429 .57 SEC14-like 2(S. cerevisiae) SEC14L2 202575_at NM_001878 .56 cellular retinoic acidbinding protein 2 CRABP2 210397_at U73945 .52 defensin, beta 1 DEFB1205428_s_at NM_001740 .51 calbindin 2, 29 kDa (calretinin) CALB2204268_at NM_005978 .50 S100 calcium binding protein A2 S100A2214088_s_at AW080549 .49 fucosyltransferase 3 FUT3 214599_at NM_005547.49 involucrin IVL 205926_at NM_004843 .49 interleukin 27 receptor,alpha IL27RA 219578_s_at AF329403 .48 cytoplasmic polyadenylationelement BP 1 CPEB1 216379_x_at AK000168 .43 CD24 antigen CD24221577_x_at BC000529 .41 growth differentiation factor 15 GDF15213316_at AL050154 .40 KIAA1462 KIAA1462 205185_at NM_006846 .39 serineprotease inhibitor, Kazal type 5 SPINK5 219956_at NM_007210 .38UDP-N-acetyl-alpha-D-galactosamine GALNT6 212715_s_at AB020626 .27microtubule associated monoxygenase MICAL3 218963_s_at NM_015515 .26keratin 23 (histone deacetylase inducible) KRT23 206143_at NM_0001112.29 solute carrier family 26, member 3 SLC26A3 206214_at NM_005084 2.27phospholipase A2, group VII (PAF acetylhydrolase) PLA2G7 206623_atNM_000440 2.13 phosphodiesterase 6A, cGMP-specific, rod, alpha PDE6A206100_at NM_001874 1.55 carboxypeptidase M CPM 207030_s_at NM_0013211.50 cysteine and glycine-rich protein 2 CSRP2

TABLE VI Significantly Changed genes in SEB-1 sebocytes after 72 hrs13-cis RA Probe ID Accession # Fold Δ Gene Title Symbol 1042_at U2718512.25 retinoic acid receptor responder RARRES1 (tazarotene induced) 133505_at AI887421 9.89 retinoic acid receptor responder RARRES1(tazarotene induced) 1 32821_at AI762213 7.04 lipocalin 2 (oncogene24p3) LCN2 38631_at M92357 5.95 tumor necrosis factor, alpha-inducedTNFAIP2 protein 2 32570_at L76465 5.91 hydroxyprostaglandindehydrogenase 15- HPGD (NAD) 36105_at M18728 4.98 carcinoembryonicantigen-related cell CEACAM6 adhesion molecule 6 (non-specific crossreacting antigen) 40071_at U03688 4.64 cytochrome P450, family 1,subfamily B, CYP1B1 polypeptide 1 859_at U03688 4.30 cytochrome P450,family 1, subfamily B, CYP1B1 polypeptide 1 37322_s_at X82460 4.25hydroxyprostaglandin dehydrogenase 15- HPGD (NAD) 1715_at U37518 4.18tumor necrosis factor (ligand) superfamily, TNFSF10 member 10 1343_s_atS66896 3.70 serine (or cysteine) proteinase inhibitor, SERPINB3 clade B(ovalbumin), member 3 873_at M26679 3.65 homeo box A5 HOXA5 988_atX16354 3.52 carcinoembryonic antigen-related cell CEACAM1 adhesionmolecule 1 (biliary glycoprotein) 1586_at M35878 3.43 insulin-likegrowth factor binding protein 3 IGFBP3 36686_at U07919 3.29 aldehydedehydrogenase 1 family, ALDH1A3 member A3 33236_at AF060228 3.22retinoic acid receptor responder RARRES3 (tazarotene induced) 3 37233_atAF079167 3.08 oxidised low density lipoprotein (lectin-like) OLR1receptor 1 36609_at D26443 3.06 solute carrier family 1 (glial highaffinity SLC1A3 glutamate transporter), member 3 1890_at AB000584 3.00growth differentiation factor 15 GDF15 33131_at X70683 2.98 SRY (sexdetermining region Y)-box 4 SOX4 35064_at X81006 2.74 tripartitemotif-containing 31 TRIM31 2031_s_at U03106 2.60 cyclin-dependent kinaseinhibitor 1A (p21, CDKN1A Cip1) 40445_at AF017307 2.51 E74-like factor 3(ets domain transcription ELF3 factor, epithelial-specific) 37821_atAF041260 2.48 breast carcinoma amplified sequence 1 BCAS1 37533_r_atD86980 2.46 tetratricopeptide repeat domain 9 TTC9 669_s_at L05072 2.42interferon regulatory factor 1 IRF1 38584_at AF026939 2.42interferon-induced protein with IFIT3 tetratricopeptide repeats 335029_at Y07828 2.35 tripartite motif-containing 31 TRIM31 41814_atM29877 2.29 fucosidase, alpha-L- 1, tissue FUCA1 909_g_at M14660 2.23interferon-induced protein with IFIT2 tetratricopeptide repeats 236634_at U72649 2.20 BTG family, member 2 BTG2 908_at M14660 2.20interferon-induced protein with IFIT2 tetratricopeptide repeats 21582_at M29540 2.18 carcinoembryonic antigen-related cell CEACAM5adhesion molecule 5 384_at X71874 2.09 proteasome (prosome, macropain)PSMB10 subunit, beta type, 10 41433_at M73255 2.08 vascular celladhesion molecule 1 VCAM1 39669_at AJ009985 2.07 annexin A9 ANXA938389_at X04371 2.07 2′,5′-oligoadenylate synthetase 1, OAS1 40/46 kDa1882_g_at HG4058-HT4328 2.06 — — 33559_at U61412 1.94 PTK6 proteintyrosine kinase 6 PTK6 40511_at X58072 1.85 GATA binding protein 3 GATA338388_at M11810 1.82 2′,5′-oligoadenylate synthetase 1, OAS1 40/46 kDa38042_at X03674 1.79 protein kinase C, alpha PRKCA 902_at L41939 1.78EPH receptor B2 EPHB2 34162_at D84111 1.78 RNA binding protein withmultiple splicing RBPMS 38269_at AL050147 1.77 protein kinase D2 PRKD238242_at AF068180 1.76 B-cell linker BLNK 37383_f_at X58536 1.71 majorhistocompatibility complex, class I, HLA-B /// HLA-C B /// majorhistocompatibility complex, class I, C 39665_at U33267 1.71 glycinereceptor, beta GLRB 881_at M35198 1.71 integrin, beta 6 ITGB6 544_atS76638 1.70 nuclear factor of kappa light polypeptide NFKB2 geneenhancer in B-cells 2 (p49/p100) 37643_at X63717 1.69 Fas (TNF receptorsuperfamily, member 6) FAS 33267_at AF035315 1.66 ATPase,aminophospholipid transporter ATP8A1 (APLT), Class I, type 8A, member 11562_g_at U27193 1.66 dual specificity phosphatase 8 DUSP8 37451_atAL109695 1.62 solute carrier organic anion transporter SLCO3A1 family,member 3A1 35194_at X53463 1.60 glutathione peroxidase 2(gastrointestinal) GPX2 34213_at AB020676 1.59 KIBRA protein KIBRA40898_at U46751 1.56 sequestosome 1 SQSTM1 322_at D88532 1.47phosphoinositide-3-kinase, regulatory PIK3R3 subunit 3 (p55, gamma)39015_f_at L42611 −4.76 keratin 6A /// keratin 6C KRT6A KRT6C 34721_atU42031 −4.00 FK506 binding protein 5 FKBP5 39016_r_at L42611 −3.85keratin 6A /// keratin 6C KRT6A KRT6C 33821_at AL034374 −3.23 ELOVLfamily member 5, elongation of ELOVL5 long chain fatty acids (FEN1/Elo2,SUR4/Elo3-like, yeast) 1610_s_at J00139 −3.12 dihydrofolate reductaseDHFR 32005_at M57703 −2.78 pro-melanin-concentrating hormone PMCH39681_at AF060568 −2.70 zinc finger and BTB domain containing 16 ZBTB1636739_at U54617 −2.56 pyruvate dehydrogenase kinase, PDK4 isoenzyme 431691_g_at U08997 −2.50 glutamate dehydrogenase 1 GLUD1 36922_at X59618−2.27 ribonucleotide reductase M2 polypeptide RRM2 32251_at AA149307−2.27 transcription elongation factor A (SII)-like 4 TCEAL4 36270_atU04343 −2.13 CD86 antigen (CD28 antigen ligand 2, B7- CD86 2 antigen)39677_at D80008 −2.04 DNA replication complex GINS protein PSF1 PSF140137_at M31724 −2.04 protein tyrosine phosphatase, non-receptor PTPN1type 1 34944_at U51704 −2.04 — — 39153_r_at U06632 −1.96 coilin COIL779_at D21337 −1.92 collagen, type IV, alpha 6 COL4A6 37062_at S62907−1.92 gamma-aminobutyric acid (GABA) A GABRA2 receptor, alpha 2 32283_atAI239869 −1.89 Iduronate 2-sulfatase MPS2 39338_at AI201310 −1.82 S100calcium binding protein A10 (annexin S100A10 II ligand, calpactin I,light polypeptide (p11)) 115_at X14787 −1.79 thrombospondin 1 THBS136104_at AA526497 −1.79 ubiquinol-cytochrome c reductase hinge UQCRHprotein 35938_at M72393 −1.79 phospholipase A2, group IVA (cytosolic,PLA2G4A calcium-dependent) 35342_at AF052159 −1.72 protein tyrosinephosphatase-like (proline PTPLB instead of catalytic arginine), member b1536_at U77949 −1.64 CDC6 cell division cycle 6 homolog CDC6 (S.cerevisiae) 41547_at AF047472 −1.59 BUB3 budding uninhibited by BUB3benzimidazoles 3 homolog (yeast) 41529_g_at W72239 −1.49 hypotheticalprotein LOC130074 LOC130074

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Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A method of treating retinoid-responsive condition in a subject,comprising: administering a therapeutically effective amount of aprotein selected from: a protein comprising the amino acid of SEQ ID No.1; a protein comprising the amino acid of SEQ ID No. 2; a proteinencoded by the complement of a nucleic acid that hybridizes under highlystringent conditions with the nucleotide sequence of SEQ ID No. 3; aprotein encoded by the complement of a nucleic acid that hybridizesunder highly stringent conditions with the nucleotide sequence of SEQ IDNo. 4; wherein the highly stringent conditions are: hybridization in asolution containing 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes;a protein comprising an amino acid sequence that is at least 95%identical to SEQ ID No. 1; and a protein comprising an amino acidsequence that is at least 95% identical to SEQ ID No. 2, to a subjecthaving a retinoid-responsive condition.
 2. The method of claim 1,wherein the retinoid-responsive condition is selected from the groupconsisting of: acne, rosacea, psoriasis, promyelocytic leukemia andneuroblastoma
 3. The method of claim 1, wherein the protein isadministered to skin affected by a retinoid-responsive conditionselected from the group consisting of: acne, rosacea and psoriasis. 4.The method of claim 1, wherein the protein is administered to skinaffected by acne in the subject.
 5. A method of treating aretinoid-responsive condition in a subject, comprising: administering atherapeutically effective amount of a substance selected from the groupconsisting of: neutrophil gelatinase-associated lipocalin (NGAL), anNGAL analog, an NGAL stimulator and an analog of an NGAL stimulator, toa subject having a retinoid-responsive condition.
 6. The method of claim5, wherein the NGAL analog is an NGAL receptor agonist.
 7. The method ofclaim 5, wherein the NGAL is encoded by a nucleic acid in an expressionvector.
 8. The method of claim 5, wherein the NGAL stimulator isselected from the group consisting of: a Toll-like receptor 2 ligand, aToll-like receptor 3 ligand, an activator of NFκB, leukotriene B4,cholesterol oleate and IL-17.
 9. The method of claim 5, wherein theactivator of NFκB is selected from the group consisting of: IL1-beta andTNF-alpha.
 10. The method of claim 8, wherein the Toll-like receptor 2ligand isN-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine.11. The method of claim 8, wherein the Toll-like receptor 3 ligand ispolyinosinic:polycytidylic acid.
 12. The method of claim 5, wherein theretinoid-responsive condition is selected from the group consisting of:acne, rosacea, psoriasis, promyelocytic leukemia and neuroblastoma. 13.The method of claim 5, wherein the substance is administered to skinaffected by a retinoid-responsive condition selected from the groupconsisting of: acne, rosacea and psoriasis.
 14. The method of claim 5,wherein the substance is administered to skin affected by acne in thesubject.
 15. A method of treating a retinoid-responsive condition in asubject, comprising: stimulating NGAL expression in a subject havingacne.
 16. The method of treating a retinoid-responsive condition in asubject of claim 15, wherein NGAL expression is stimulated byadministration of an NGAL stimulator which upregulates transcription ofa gene encoding NGAL.
 17. The method of treating acne in a subject ofclaim 15, wherein NGAL expression is stimulated by administration of anucleic acid encoding NGAL to the subject.
 18. The method of claim 16,wherein the NGAL stimulator is selected from the group consisting of: aToll-like receptor 2 ligand, a Toll-like receptor 3 ligand, an activatorof NFκB, leukotriene B4, cholesterol oleate and IL-17.
 19. The method ofclaim 18, wherein the activator of NFκB is selected from the groupconsisting of: IL1-beta and TNF-alpha.
 20. The method of claim 18,wherein the Toll-like receptor 2 ligand isN-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine.21. The method of claim 18, wherein the Toll-like receptor 3 ligand ispolyinosinic:polycytidylic acid.
 22. A method of inhibitingPropionibacterium acnes, comprising: contacting a Propionibacteriumacnes bacterium with a substance selected from the group consisting of:NGAL and an NGAL analog.
 23. The method of inhibiting Propionibacteriumacnes of claim 22, wherein the Propionibacterium acnes bacterium is incontact with a human subject.
 24. The method of inhibitingPropionibacterium acnes of claim 23, wherein the human subject has acne.25. The method of inhibiting Propionibacterium acnes of claim 23,wherein the human subject has rosacea.
 26. A composition for treating aretinoid-responsive condition in a subject, comprising a substanceselected from the group consisting of: neutrophil gelatinase-associatedlipocalin (NGAL), an NGAL analog, an NGAL stimulator, an analog of anNGAL stimulator and a combination of two or more of: neutrophilgelatinase-associated lipocalin (NGAL), an NGAL analog, an NGALstimulator and an analog of an NGAL stimulator.
 27. The composition ofclaim 26, wherein the NGAL stimulator is selected from the groupconsisting of: a Toll-like receptor 2 ligand, a Toll-like receptor 3ligand, an activator of NFκB,N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine,polyinosinic:polycytidylic acid, leukotriene B4, cholesterol oleate,IL-17 and a combination of two or more of a Toll-like receptor 2 ligand,a Toll-like receptor 3 ligand, an activator of NFκB,N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine,polyinosinic:polycytidylic acid, leukotriene B4, cholesterol oleate andIL-17.
 28. The composition of claim 26, wherein the substance isselected from: a protein comprising the amino acid of SEQ ID No. 1; aprotein comprising the amino acid of SEQ ID No. 2; a protein encoded bythe complement of a nucleic acid that hybridizes under highly stringentconditions with the nucleotide sequence of SEQ ID No. 3; a proteinencoded by the complement of a nucleic acid that hybridizes under highlystringent conditions with the nucleotide sequence of SEQ ID No. 4;wherein the highly stringent conditions are: hybridization in a solutioncontaining 6×SSC, 5×Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1×SSC and 0.1% SDS at 60° C. for 15 minutes;a protein comprising an amino acid sequence that is at least 95%identical to SEQ ID No. 1; and a protein comprising an amino acidsequence that is at least 95% identical to SEQ ID No. 2, to a subjecthaving a retinoid-responsive condition.